The invention relates to methods of identification or evaluation of compounds useful in the field of fragrances and aromas (the volatile parts of flavour). Methods according to the invention take into account the occurrence of enzymatic metabolism in the human respiratory tract, including the oral and in particular the nasal cavity.
Compounds which reach the nasal cavity and exert a particular effect, such as binding to receptors, which is a prerequisite to perceive olfactory stimuli, are constantly changing their environment and different physico-chemical specificities are of advantage and disadvantage in each phase of their lifetime. First, the compound is in a base, e.g. as part of a fragrance oil where a particular vapour pressure is required to be volatile. Depending on the type of odour release (directly transfer in headspace, or dispersion, e.g. as aerosols) different properties are of advantage for the constituents of the perfume mixture. While a high vapour pressure is often desirable, the compound has to be able to readily dissolve into the nasal mucosal fluid covering the neuronal cells. Finally, the compound needs to bind and activate receptor proteins. For most of the journey from base to receptor, the odorant compounds appear to remain unchanged. However, the situation seems to be more complicated. It has been speculated that metabolism is rendering odorant compounds inactive to make them more water-soluble and facilitate clearance from the nasal epithelium.
Furthermore, it has been speculated that, for some fragrance ingredients, the compound that is directly responsible for the perception of the odour (the odorant), is not the fragrance ingredient itself. Instead, the fragrance ingredient may merely be a non-odorant precursor that forms, as a metabolite, the actual odorant which activates the olfactory receptor to result in olfactory perception. Said odorant metabolites may be formed enzymatically in the human respiratory tract, particularly in the epithelium of the human nose.
Metabolism of such precursors that are substrates of enzymes may occur prior to receptor binding in the fluidic mucus or in cells lining the cavity or it may occur after receptor activation. This may change their various abilities that influence odorant perception, including its physico-chemical properties (for example solubility in mucosal fluid) and activation of the receptor. The metabolite(s) may have chemical and/or physical properties which are of advantage for interaction with receptors, other enzymes and/or odorant binding proteins. Substrates may be odorant compounds or non-odorant compounds. In the case of the latter, one or more metabolite of the substrate may be an odorant, and/or have the above-mentioned properties.
Metabolism may inactivate or activate receptor ligands. Compounds of interest may be agonists, antagonists, enzyme substrates, enzyme inhibitors, and allosteric regulators of receptors or enzymes. The metabolites may compete, for example, for receptor binding, interact with additional receptors and enzymes, and/or modulate the activity and sensitivity of receptors and enzymes. The metabolites generated from substrates of metabolic enzymes may have properties that enable them to interact with receptors and enzymes and these metabolites may in fact be primarily responsible for the perceived quality and effects of flavour and fragrance ingredients and/or compete with their substrates for receptor interaction, and in particular for receptor activation.
However, metabolism involving odorant compounds in the human respiratory tract, particularly in the olfactive mucosa, has not been shown in vivo.
The present invention employs known analytical methods to provide a method of identifying, analysing or evaluating test compounds useful in the field of fragrances and aromas.
Compounds useful in the field of fragrances and aromas may be fragrance and aroma compounds as such, but also modulators of their perception. Modulators are compounds that influence the olfactive perception of odorant compounds. A modulator may result in changes of intensity (overall enhancer or masking agent), quality (change of olfactive note, enhancing or masking of particular notes), duration/longevity of perception, or combinations of these. A modulator may enhance the overall perception of a particular odorant or mixture of odorants, or a particular olfactive quality/note. A modulator may reach these effects by modulating, influencing or regulating metabolic reactions in the respiratory tract. A modulator may enhance or suppress metabolism, for example by affecting the enzyme directly. Instead of influencing enzymes, a modulator may affect (activate or block) one or more receptors to reach an enhancing or blocking/masking effect, or to influence the quality and olfactive note of perception. A modulator may extend the “life-time” or duration of the effect of an odorant by hindering or suppressing its usual metabolism and thereby enhancing its intensity or longevity. A similar enhancing effect will be caused by a compound that positively influences the rate of enzymatic activity, provided that the substrate that is metabolised to form its metabolite is not odorant (or less odorant) than its odorant metabolite. Modulators may suppress metabolism occurring in the human respiratory tract and/or change the olfactive quality of single odorants.